The invention relates to the fields of insect control using baculoviruses and control of baculoviral infections in invertebrates.
Historically, mosquito control operations have primarily used chemical pesticides or the bacterium Bacillus thuringiensis israelensis (Bti) for control of larval mosquitoes. Chemical larvicides are rapidly losing market share due to safety issues and the expense of treating large aquatic habitats, where the larvae are produced. While Bti is effective and safe, it is quickly inactivated in the environment, and works poorly in polluted water habitats.
The other major biocontrol for insects is viruses, and the Baculoviridae family of viruses, commonly called baculoviruses, has been widely studied and developed commercially for certain insects. Baculoviruses are composed of large, double-stranded, circular DNA molecules that are packaged in rod-shaped capsids. The Baculoviridae includes the Nucleopolyhedroviruses and the Granuloviruses subgroups (see Granados R R and B A Federici, Eds. 1986 The Biology of Baculoviruses, Vol. 1. Boca Raton, Fla.: CRC Press; Volkman, L E et al. 1995. Baculoviruses. In: Virus Taxonomy. Sixth Report of the International Committee on Taxonomy of Viruses. Murphy, F A et al. Eds Springer Verlag Wien).
Insect control using baculoviruses is an attractive approach, because baculoviruses only infect invertebrates, and they can generally be produced in sufficiently high numbers for commercial use. The viruses are produced naturally in two forms: xe2x80x9coccludedxe2x80x9d and xe2x80x9cnon-occludedxe2x80x9d. The occluded form, in which the DNA genome is surrounded by crystalline proteins (forming a structure called the inclusion or occlusion body), is the form in which the virus is spread naturally from one insect to another. It is also the form that makes a good insecticide formulation, since it is stable in the environment and in commercial packaging, and it can be mixed more readily with other pesticide formulations.
Despite the many advantages of baculoviruses for insect control, one commercial disadvantage is that a single baculovirus species has a fairly narrow host range, so a given baculovirus cannot be used for broad spectrum insect control. One of the best studied baculoviruses, Autographa californica nuclear polyhedrosis virus (AcNPV), is considered to have a fairly broad host range for a baculovirus, since it is known to infect over thirty species of Lepidoptera. The celery looper multiple embedded virus described by Hostetter and Puttler (U.S. Pat. No. 4,911,913) also is considered to have broad insecticidal activity within the Lepidoptera.
Thus, to develop an effective baculoviral control strategy for mosquitoes, there is a need to identify a viral strain that will infect as many mosquito species as possible. The first documented report of a mosquito pathogenic virus was made in 1963 with the discovery of a xe2x80x9ccytoplasmic polyhedrosis virusxe2x80x9d from Culex tarsalis in California (Kellen et al., 1963, J. Insect Pathol. 5:98-103), but this virus was later shown to be a densonucleosis virus, from the family Parvoviridae (Federici, B A 1985, xe2x80x9cViral Pathogensxe2x80x9d. IN Biological Control of Mosquitoes, H C Chapman, Ed., Bull. No. 6, American Mosquito Control Association, Inc., pp. 62-74). Additional viruses pathogenic to mosquitoes have been reported from many different mosquito hosts, primarily by researchers in the U.S., Europe and Russia (Federici, 1985; ibid.; Goettel, M S 1985 Agr. Forestry Bull. 8:41-44). The first report of a baculovirus from a mosquito host was from Aedes sollicitans collected in Louisiana (Clark, T B, H C Chapman, and T Fukuda, 1969, J. Invert. Pathol. 14(2);284-286). Natural infections with NPVs have been reported from about ten mosquito species representing the genera Aedes, Anopheles, Culex, Psorophora, Uranotaenia and Wyeomyia (Federici, ibid.). Such infections were identified as being NPV infections based only on electron microscopic observations of the shape and size of occlusion bodies in collected larvae and on histopathological and cytopathological observations on infected larvae (Federici and Lowe 1972 Invert. Pathol. 20:14-21; Federici, B A 1980 Virology 100: 1-9; Stiles and Paschke 1980 J. Invertebr.Pathol. 35: 58-64). The AesoNPV characterized by these researchers produces spindle-shaped occlusion bodies 5-20 microns in length. H. Chapman (1974, Ann Rev Entomol 19:33-59) characterized an NPV infection in Culex quinquefasciatus in which the occlusion bodies are globular and from 1-5 microns in diameter. A viral disease of Culex tarsalis, characterized by tetragonal inclusion bodies, was described by W. R. Kellen et al. (1963, J. Insect Pathol. 5:98-103). While this virus was lost upon culturing, Clark and Chapman concluded it was the same virus that infected Culex salinarius (1969, J. Invert. Pathol 13:312), a virus that did not infect Culex peccator or Culex quinquefasciatus. 
This area of mosquito pathology has received very little attention since these preliminary, initial reports 25-30 years ago, despite the tremendous advancements made with other insect baculoviruses for use as biopesticides and expression vectors (Adams, J R and J T McClintock, 1991, pp. 87-204 In: Atlas of Invertebrate Viruses, Adams and Bonami, Eds., Boca Raton: CRC Press; Possee, R D 1997, Curr. Opin. Biotechnol. 8: 569-572; and Possee, R D et al. 1997 Pestic. Sci. 51: 462-470), and no NPV has been purified or isolated from mosquitoes to allow molecular studies for unequivocal identification. A major reason for the inactivity in this area has centered on the inability to reliably transmit mosquito baculoviruses to their original and alternate hosts (Federici, 1985). There are numerous factors that can affect the infectivity of a baculovirus for an insect host, including chemical, physical and/or biological factors.
Much effort has been directed at developing methods and protocols to enhance the storage and stability of baculoviruses (usually in occluded form), for use in field applications. Factors that either reduce or enhance infectivity have been identified as components of storage mixtures, formulations, or crops on which baculoviral applications are to be made. For example, several proteins have been shown to enhance baculoviral infections. Enhancin, a metalloproteinase, has been isolated from an armyworm (Trichoplusia ni) granulosis virus (GV), and it can enhance the activity of baculoviruses by digesting certain peritrophic membrane proteins (Corsaro et al. 1993. In xe2x80x9cParasites and Pathogens of Insectsxe2x80x9d, Beckage, N E et al., Eds., Volume 2: pp. 127-145, Academic Press, San Diego; Lepore L S. et al. 1996 J. Invertebr. Pathol. 68(2): 131-140; Wang P and R R Granados 1997, Proc. Natl. Acad. Sci. USA 94(13): 6977-6982; Wang, P. et al. 1997 J. Gen. Virol. 78: 3081-3089). A virus enhancing protein has also been detected in the spheroid, spindle and virion of an entomopoxvirus (Wijonarko A and T Hukuhara 1998, J. Invertebr. Pathol. 72(1): 82-86). Chitinase has also been shown to enhance infectivity of the gypsy moth NPV (Shapiro M et al. 1987, J. Econ. Entomol. 80(6): 1113-1116). Several acids, including boric and sorbic, have been shown to enhance infectivity of lepidopterous baculoviruses (Morales L et al. 1997, Ann. Soc. Entomol. Bras. 26(1): 115-120; Shapiro M and R A Bell 1982, Ann. Entomol. Soc. Am 75(3):346-349), while tannic acids have been shown to inhibit NPV infectivity (Keating et al. 1989, J. Invertebr. Pathol. 54(2):165-174; Young et al. 1995, Biological Control 5(2): 145-150).
Studies investigating the effects of various plant constituents on the infectivity of baculoviruses found that most of these actually inhibit infectivity (Elleman C J and Entwistle P F 1985, Ann. Appl. Biol 106(1): 83-92, 93-100; Felton G W and S S Duffey 1990, J. Chem. Ecol 16(4): 1221-1236; Hoover K et al. 1998 J. Chem. Ecol. 24(2): 253-271; Keating, S T et al., 1990 J. Invertebr. Pathol. 56(3): 317-326).
In addition to the use of naturally occurring baculoviruses as insect control agents, it is possible to improve the insecticidal capabilities of such baculoviruses by engineering the virus so that it is more aggressive in killing its host, which is not typically the goal of a natural viral infection. By improving the rate of killing, the engineered baculoviruses limit the feeding and consequently damage caused to crops. Since farmers tend to apply insecticides only when damage becomes visible, it is important that the insecticide act swiftly. For example, Miller and O""Reilly have described a mechanism of improving the LT50 (the time needed to kill 50% of the insect population) by deleting a gene which causes the production of an enzyme that inactivates insect molting hormones, ecdysteroid UDP-glucosyl transferase (EGT) (U.S. Pat. No. 5,352,451). The expression of the baculovirus EGT gene in wild-type baculoviruses prevents the insect larva from molting or pupating, allowing more time for the virus to propagate itself in the larval stage. Inactivation of the baculovirus EGT gene allows molting and pupation of the infected larva to proceed, which results in a shorter larval stage and less damage to crops. As another example, Hammock et al. (U.S. Pat. No. 5,674,485) have shown that juvenile hormone esterase can also be used in recombinant baculoviral insecticides to improve the LT50.
Several authors have described techniques and methods for engineering insect toxin protein genes into the baculoviral genome, providing for exposure of the insect to not only an infectious virus particle but additionally to a debilitating toxic molecule, such as polypeptide toxins produced by scorpions, wasps, mites, or spiders. Tomalski et al., U.S. Pat. No. 5,266,317, issued Nov. 30, 1993, describe the use of recombinant baculoviruses that express an insect-specific paralytic neurotoxin of an insect predacious mite. Additionally, insect-selective toxins have been identified in venoms from snails, spiders, and a number of other arthropods (see review by Zlotkin, 1985, In Comprehensive Insect Physiology, Biochemistry, and Pharmacology, Vol. 10, Chapter 15, pp. 499-541). The use of more than one toxin gene in a recombinant baculovirus has also been described, to maximize the insect-killing rate and to minimize the chance for resistance to develop (U.S. Pat. No. 5,756,340 Hammock, et al.).
There remains a need to identify methods for effective transmission of baculoviruses to mosquitoes in the environment. There also remains a need to provide improved baculovirus compositions for enhanced transmission of baculoviruses to insects in the environment.
This invention provides an insecticidal composition comprising (a) a baculovirus effective for producing a lethal or debilitating infection in an insect; and (b) at least 1 mM of a divalent cation selected from the group consisting of: magnesium, cobalt, strontium and nickel is disclosed. Also provided are methods for controlling insect pests comprising the step of applying an insect-controlling amount of the composition to a habitat for the insect pests.
This invention further provides novel, isolated mosquito-infecting baculoviruses having at least one of the following characteristics: (a) nuclear occlusion bodies, globular in shape, diameter approximately 400 nm, each containing between 1-8 rod-shaped virions, approximately 200xc3x9740 nm; (b) nuclear occlusion bodies with no surrounding membrane and with a density of approximately 1.14-1.18 g/ml which agglutinate upon freezing; and (c) infectious in Culex quinquefasciatus and Culex nigripalpus. The novel mosquito baculoviruses of this invention can also be characterized by nucleic acid sequences provided herein, as well as by polypeptides encoded by the nucleic acid sequences. Methods for the in vitro propagation of mosquito baculoviruses comprising infecting a culture of mosquito cells with the mosquito baculovirus, growing the cells in culture medium containing at least 1 mM of a divalent cation selected from the group consisting of: magnesium, cobalt, strontium and nickel and a chelator or binder that selectively reduces the effective calcium ion concentration below 1 mM until inclusion bodies are detected in the cells, and harvesting the virus from the cells, are also provided.
Other aspects of the invention comprise isolated nucleic acids of the novel mosquito baculoviruses, isolated nucleic acids encoding polypeptides of the novel mosquito baculovirus, and cells containing these nucleic acids.
Also provided are methods for controlling baculovirus infections in invertebrates comprising administering to invertebrates calcium, iron or copper ions to provide an effective concentration of at least 1 mM.
Definitions
xe2x80x9cBaculovirusxe2x80x9d means a member of the family Baculoviridae, whether currently known or described in the future.
xe2x80x9cCuniNPVxe2x80x9d means a novel baculovirus that infects Culex nigripalpus. 
xe2x80x9cInsect-antagonistic genexe2x80x9d means any gene, the product (which may be anti-sense nucleic acid or a polypeptide) of which interferes with the growth, development, or reproduction of an insect. Examples of insect-antagonistic genes include toxins, insect hormones, insect hormone mimics, and sterilization peptides.
Detailed Description
Described herein is the discovery of improved insecticidal compositions for the control of insects using viruses of the Baculoviridae. The invention described herein includes a baculovirus-based insecticidal composition comprising (a) a baculovirus effective for producing a lethal or debilitating infection in an insect, and (b) at least 1 mM of a divalent cation selected from the group consisting of: magnesium, cobalt, strontium and nickel. In a preferred embodiment, the composition further comprises an insecticidal composition-suitable carrier. The baculovirus of the invention is any virus of the family Baculoviridae, previously known or hereafter isolated and described. As currently classified, the Baculoviridae consists of double-stranded, circular, closed DNA viral genomes (between approximately 80 and 230 kilobases) that are typically packaged in rod-shaped capsids, with a general size range of approximately 30-60 nanometers by 250-300 nanometers (Miller, L 1996 xe2x80x9cInsect Virusesxe2x80x9d In Fundamental Virology, 3rd Ed., Fields B N et al eds., Lippincott-Raven Publ., Philadelphia, Pa. USA). The existing genera and species in the Baculoviridae are listed in Virus Taxonomyxe2x80x946th Report of the International Committee on Taxonomy of Viruses, Murphy F A et al. eds., Springer Verlag, Wien, 1995, pp 104-111. Baculoviruses are currently known to infect insects of the Lepidoptera, Coleopterra, Hymenoptera and Diptera, including but not limited to Autographa californica, Heliothis zea, Heliothis armigera, Syngrapha falcifera, Spodoptera frugiperda, and Thichoplusia ni (a complete list of hosts for known baculoviruses is provided in the Virus Taxonomy reference cited above). New members of the Baculoviridae are continually being discovered, and the compositions and methods of this invention are fully applicable to baculoviruses discovered in the future.
A lethal or debilitating infection in an insect is an infection in the larval stage which prevents the majority of adults from emerging, or in the cases where the adult emerges, in which the adult reproductive capacity is statistically significantly diminished. The divalent cation to be included in the composition can be Mg++. Alternatively, Co++, Sr++ or Ni++ can be used. In a preferred embodiment in a liquid formulation, at least 5 mM Mg++ is used. No upper limit of the divalent cation, e.g. Mg++ need be specified so long as the amount used is not toxic. For example, between 5 and 40 mM Mg++ can be used. Alternatively, a combination of divalent ions comprising Mg++ and one or more of the ions Co++, Sr++ or Ni++, giving a total divalent concentration of at least 1 mM and preferably at least 5 mM, can be used. When used in a non-liquid formulation, the amount of divalent cation should be calculated on a weight/weight basis, i.e. 5 mM MgCl2 would be approximately 1 g MgCl2 per kg or liter of the formulation.
In a specific embodiment, the baculovirus-based insecticidal composition further comprises a chelator or binder that selectively reduces the effective calcium ion concentration below approximately 1 mM. In specific embodiments, the chelator or binder reduces the effective calcium ion concentration below approximately 0.5 mM or below 0.1 mM.
In another embodiment, the invention is a mosquito larvacidal composition comprising (a) a baculovirus effective for producing a lethal or debilitating infection in mosquito larvae; and (b) at least 1 mM of a divalent cation selected from the group consisting of: magnesium, cobalt, strontium and nickel. In a preferred embodiment, the composition further comprises a larvacidal composition-suitable carrier.
In preferred embodiments, the mosquito larvacidal composition comprises a baculovirus selected from the group of nuclear polyhedrosis viruses that infect Culex, Aedes, Anopheles, Psorophoia, Uranotaenia, and Wyeomyia mosquito species. In specific embodiments, the baculovirus can be AesoNPV or the novel baculovirus CuniNPV further described hereinbelow. In a specific embodiment, the composition is a preparation of air-dried, baculovirus-infected mosquito larvae that are processed into a powder containing at least 1 mM of a divalent cation selected from the group consisting of: magnesium, cobalt, strontium and nickel.
In other embodiments, the mosquito larvacidal composition further comprises a chelator or binder that selectively reduces the effective calcium ion concentration below approximately 1 mM. In specific embodiments, the chelator or binder reduces the effective calcium ion concentration below approximately 0.5 mM or below 0.1 mM.
Given the effectiveness of divalent cations, particularly Mg++, in combination with the novel mosquito baculovirus of this invention, in enhancing baculovirus infectivity of mosquitos, it is possible to use this combination to enhance the infectivity of other agents, e.g. bacteria or other viruses, in mosquitos. Thus, in another embodiment, the mosquito larvacidal composition comprises (a) a microorganism or virus capable of producing a lethal or debilitating infection in mosquito larvae; (b) a mosquito baculovirus; and (c) at least 1 mM of a divalent cation selected from the group consisting of: magnesium, cobalt, strontium and nickel. In specific embodiments, the microorganism can be a bacterium or a fungus, the virus can be any mosquito-infecting virus, and the mosquito baculovirus is CuniNPV (as described herein). In a further embodiment, the composition further comprises a larvacidal composition-suitable carrier. In other embodiments, the mosquito larvacidal composition further comprises a chelator or binder that selectively reduces the effective calcium ion concentration below approximately 1 mM. In specific embodiments, the chelator or binder reduces the effective calcium ion concentration below approximately 0.5 mM or below 0.1 mM.
Generally, the insecticidal or larvacidal compositions can be in the form of: wettable powders, dispersible granular formulations, granules, suspensions, emulsions, solutions for aerosols, baits and other conventional insecticide preparations. Carriers for insecticidal compositions are well-known in the art and one of skill in the art can choose an appropriate carrier based on the application site and the target insect. Examples of techniques for the formulation of baculoviruses as insecticidal compositions are described in Rhodes D J (1993, xe2x80x9cFormulation of Biological Control Agentsxe2x80x9d In Exploitation of Microorganisms; Jones D G ed., Chapman and Hall, London, pp. 411-429) and references cited therein. The insecticidal composition-suitable carrier can be a solid or liquid diluent or carrier. The diluent or carrier can be a liquid, such as water, alcohol, hydrocarbons or other organic solvents, or a mineral, animal or vegetable oil, or a solid, such as a powder, e.g. talc, clay, silicate or corn cob grits. Wetting agents, coating agents, agents to promote physical flexibility, UV protectants, dispersants and sticking agents can also be added to the insecticidal compositions of the present invention. Similarly, nutrients may be added to increase feeding behavior and/or to attract insects. Flow agents, for example, clay-based flow agents, may be added to minimize caking of the wettable powders or other dry preparations during storage. Alternatively, the insecticidal or larvacidal composition can be microencapsulated using plant lignins or other suitable microencapsulating materials as the carrier.
In any insecticidal or larvacidal composition of the invention, the baculovirus can be a recombinant baculovirus that has been engineered to express one or more insect-antagonistic genes. The insect-antagonistic gene can be any gene that interferes with the growth, development or reproduction of the insect. Such an engineered baculovirus is more aggressive in killing its host. By improving the rate of killing, such engineered baculoviruses may limit the number of subsequent generations of the insect that are produced, limit the opportunity for disease-causing viruses vectored by the insect to increase in number, and limit the potential for insects to develop resistance to the gene. In specific embodiments, the insect-antagonistic gene is selected from the group consisting of: scorpion toxins, straw itch-mite toxin, spider toxins, snail toxins, insect hormones, insect hormone mimics, sterilization peptides. However, it is recognized that it may not always be advantageous to improve the rate of killing of the insect. For example, in the case of mosquito larvae, a slower kill rate may be advantageous because it maintains competition for nutrients and space against noninfected larvae.
Recombinant baculoviruses can be made using techniques well-established in the art. Generally, the recombinant baculovirus may be prepared by cloning a gene encoding the insect-antagonistic gene into a baculovirus transfer vector at a restriction site downstream of a promoter capable of directing expression of the insect-antagonistic gene in insect cells, and co-transfecting cells susceptible to baculovirus infection with the recombinant transfer vector and an intact wild-type baculovirus DNA. Homologous recombination occurs, resulting in a recombinant baculovirus harbouring the chimeric gene operably linked to a promoter (U.S. Pat. No. 5,166,317). The recombinant baculovirus may or may not contain an expressible polyhedrin gene. When an expressible polyhedrin gene is provided, the occluded form of the virus is produced. Alternatively, those of routine skill in the art can construct recombinant, occluded baculoviruses by coinfection of cells with a helper virus which supplies polyhedrin gene function. The construction of a stable, occluded recombinant nuclear polyhedrosis virus which has a functional polyhedrin gene and which, in insect cells infected therewith, expresses an insect-antagonistic gene which is secreted by the cells, the insect-antagonistic gene product being expressed with a signal peptide such that the insect-antagonistic gene product is secreted from the insect cells is described in U.S. Pat. No. 5,770,192.
The present invention also provides methods for controlling insect pests comprising the step of applying an insect-controlling amount of the insecticidal compositions of this invention to a habitat for the insect pest. The insect-controlling amount is the amount sufficient to interfere with the growth, development and/or reproduction of the insect. The insect controlling amount can readily be determined experimentally by a person of ordinary skill in the art using bioassays and other information available in the art, examples of which are described herein.
Also disclosed and claimed herein are specific methods for controlling mosquitoes comprising the step of applying a mosquito-controlling amount of a mosquito larvacidal composition to a habitat of mosquitoes. The amount to be applied can be in a liquid or non-liquid formulation. The formulation is chosen to maximize exposure of the target mosquito population to the larvacidal composition by conventional application methods. Since mosquito larvae are filter feeders, the method of delivery is by ingestion. Delivery can be enhanced by the addition of feeding stimulants. In a preferred embodiment, the virus and sufficient Mg++ are microencapsulated for delivery together to the mosquito gut. Encapsulation methods are well-documented in the art. Encapsulation of baculoviral-based insecticides helps to maintain activity by protecting the virus from harmful environmental conditions. Starch, flour and gluten have been studied extensively as materials to encapsulate insecticides. Such encapsulated insecticides can be applied as granular or sprayable formulations. Shasha et al. (U.S. Pat. No. 5,750,467) describe dispersible encapsulations or adjuvants that use plant lignins to deliver insecticides, such as baculoviruses. Lebo, Jr. et al. (U.S. Pat. No. 5,552,149) describe methods for microencapsulation such as pesticides that employ lignosulfonates, especially to provide protection from ultraviolet rays. The mosquito-controlling amount is the amount sufficient to interfere with the growth, development and/or reproduction of the mosquito larvae. In a specific embodiment, the habitat to which the larvacidal composition is applied is a mosquito breeding area, e.g. a body of water. In a specific embodiment, the mosquitoes to be controlled are members of the Culicidae.
The invention herein described further comprises a newly isolated mosquito-infecting baculovirus, hereinafter referred to as a CuniNPV, that is characterized by one or more of the following:
(a) nuclear occlusion bodies, globular in shape, diameter approximately 400 nm (e.g. xc2x110 nm), each containing between 1-8 rod-shaped virions, approximately 200xc3x9740 nm;
(b) nuclear occlusion bodies, with no surrounding membrane, with a density of approximately 1.14-1.18 g/ml which agglutinate upon freezing, and
(c) infectious in Culex quinquefasciatus and Culex nigripalpus. 
The isolated mosquito-infecting baculovirus can be further characterized by the presence of major occlusion body proteins, sized as 29.7 kDa and 97 kDa on SDS-PAGE.
Alternatively, the novel mosquito-infecting baculovirus of this invention can be characterized as a baculovirus which contains nucleic acid that shares at least 60% identity with the sequence of one or more nucleic acids selected from the group of sequences set forth as SEQ ID NO:1; SEQ ID NO:4; SEQ ID NO:10; SEQ ID NO:19; SEQ ID NO:38; SEQ ID NO:42; SEQ ID NO:44; SEQ ID NO:46; SEQ ID NO:48; SEQ ID NO:50; SEQ ID NO:53; SEQ ID NO:61; SEQ ID NO:66; SEQ ID NO:70; SEQ ID NO:72; SEQ ID NO:74; SEQ ID NO:76; SEQ ID NO:80; SEQ ID NO:83; SEQ ID NO:87; SEQ ID NO:94; SEQ ID NO:106; SEQ ID NO:117; SEQ ID NO:130; SEQ ID NO:145, and SEQ ID NO:146. In preferred embodiments, the novel mosquito-infecting baculovirus of this invention can be characterized as a baculovirus which contains nucleic acid that shares at least 70%, 80% or 90% identity with the sequence of one or more nucleic acids selected from the group of sequences set forth as SEQ ID NO:1; SEQ ID NO:4; SEQ ID NO:10; SEQ ID NO:19; SEQ ID NO:38; SEQ ID NO:42; SEQ ID NO:44; SEQ ID NO:46; SEQ ID NO:48; SEQ ID NO:50; SEQ ID NO:53; SEQ ID NO:61; SEQ ID NO:66; SEQ ID NO:70; SEQ ID NO:72; SEQ ID NO:74; SEQ ID NO:76; SEQ ID NO:80; SEQ ID NO:83; SEQ ID NO:87; SEQ ID NO:94; SEQ ID NO:106; SEQ ID NO:117; SEQ ID NO:130; SEQ ID NO:145, and SEQ ID NO:146. In specific embodiments, mosquito-infecting baculoviruses of this invention comprise a nucleic acid sequence with at least 75%, 77%, 79%, 81%, 83%, 85%, 87%, 89%, 91%, 93%, 95%, 97% or 99% identity to a nucleic acid selected from the group of sequences set forth as SEQ ID NO:147 and SEQ ID NO:148.
An example of a CuniNPV has been deposited with the American Type Culture Collection (10801 University Boulevard, P.O. Box 1549, Manassas, Va. 20108) under the Budapest Treaty on the International Recognition of the Deposit of Microorganism for the Purposes of Patent Procedure, on Feb. 3, 1999 as accession number VR-2639. The genome sequence of VR-2639 is deposited in GenBank under Accession Number AF403738.
An isolated mosquito-infecting baculovirus is one which has been either purified from infected mosquito larvae, cloned through plaque purification in mosquito cell culture, or otherwise prepared from a single viral isolate. A method for isolating the novel baculoviruses of this invention from infected mosquito larvae is described in the Examples.
Additional mosquito-infecting baculoviruses of this invention can be identified by probing viral DNA isolated from diseased mosquito larvae, obtained as herein described. One method for detection is the use of polymerase chain reaction amplification. Polymerase chain reaction (PCR) amplification can be used to detect other mosquito-infecting baculoviruses of this invention by amplifying baculoviral specific DNA sequences. Repeated cycles of denaturation, primer annealing and extension carried out with polymerase, e.g., a heat stable enzyme Taq polymerase, lead to exponential increases in the concentration of desired DNA sequences. Using the nucleic acid sequences of a mosquito baculovirus as disclosed herein, synthetic oligonucleotides, or xe2x80x9cprimersxe2x80x9d, can be prepared which are complementary to the ends of sequences which are to be amplified. The sample DNA, obtained from mosquito larval populations, can be denatured at high temperatures (e.g., 95xc2x0 C.) and then reannealed in the presence of a large molar excess of the primers. The primers, oriented with their 3xe2x80x2 ends pointing towards each other, hybridize to opposite strands of the target sequence and prime enzymatic extension along the nucleic acid template in the presence of the four deoxyribonucleotide triphosphates. The end product is then denatured again for another cycle. After this three-step cycle has been repeated several times, amplification of a DNA segment by more than one million-fold can be achieved.
Primers chosen for the PCR amplification should be at least approximately 10 nucleotides in length and amplifying a product at least approximately 30 nucleotides in length. The length and G+C content of the primers are used to determine the melting temperature (Tm) according to formulas known in the art. The Tm establishes the temperature at which primer annealing to the sample DNA is performed. For primers shorter than 20 nucleotides, an estimate of Tm can be calculated as Tm=4(G+C)+2(A+T), while for longer primers, an estimate of the Tm requires the use of the nearest neighbor calculation, which takes into account thermodynamic parameters and is embedded in most available computer programs for designing PCR primers (see, e.g. Dieffenbach, C S et al. 1995 General concepts for PCR primer design. IN: PCR Primer, A Laboratory Manual; Dieffenbach and Dveksler, eds., Cold Spring Harbor Laboratory Press, Plainview, N.Y.).
Detection of the amplified product can be by any of several standard methods, such as electrophoresis on an agarose or polyacrylamide gel and ethidium bromide staining to visualize the nucleic acids on the gel. Once the presence of a baculovirus in the sample has been detected, the novel mosquito-infecting baculovirus can be isolated and purified according to methods described herein.
The novel mosquito-infecting baculoviruses of the present invention can be propagated and produced by repeatedly infecting mosquito larvae and harvesting the larvae. Alternatively, the novel mosquito-infecting baculoviruses of the present invention can be propagated by introducing the baculovirus to a cultured mosquito cell, growing the mosquito cells for a period until inclusion bodies are detected inside the cells and then harvesting the baculovirus from the cells. Methods for culturing mosquito cells have been described (see, e.g. Kurtti and Munderloh 1984 Adv. Cell Cult. 3:259-302; Oelofsen, M J et al. 1990 J. Med. Entomol. 27:939-944; Charpentier, G et al. 1995 J. Med. Entomol. 32:793-800). In a specific embodiment, the mosquito cell culture medium for propagating the virus contains at least 1 mM divalent cation and less than 1 mM, Ca++. In a preferred embodiment, the mosquito cell culture medium contains at least 5 mM Mg++ and less than 1 mM, and preferably less than 0.5 or 0.1 mM, Ca++.
Mosquito-infecting baculoviruses of this invention with significant homology, i.e. sequence identity, to one or more nucleic acids selected from the group of sequences set forth as SEQ ID NO:1; SEQ ID NO:4; SEQ ID NO:10; SEQ ID NO:19; SEQ ID NO:38; SEQ ID NO:42; SEQ ID NO:44; SEQ ID NO:46; SEQ ID NO:48; SEQ ID NO:50; SEQ ID NO:53; SEQ ID NO:61; SEQ ID NO:66; SEQ ID NO:70; SEQ ID NO:72; SEQ ID NO:74; SEQ ID NO:76; SEQ ID NO:80; SEQ ID NO:83; SEQ ID NO:87; SEQ ID NO:94; SEQ ID NO:106; SEQ ID NO:117; SEQ ID NO:130; SEQ ID NO:145, and SEQ ID NO:146 can readily be obtained by screening collected mosquito larvae suspected of being infected with a probe comprising one or more nucleic acid sequences contained in SEQ ID NO:1; SEQ ID NO:4; SEQ ID NO:10; SEQ ID NO:19; SEQ ID NO:38; SEQ ID NO:42; SEQ ID NO:44; SEQ ID NO:46; SEQ ID NO:48; SEQ ID NO:50; SEQ ID NO:53; SEQ ID NO:61; SEQ ID NO:66; SEQ ID NO:70; SEQ ID NO:72; SEQ ID NO:74; SEQ ID NO:76; SEQ ID NO:80; SEQ ID NO:83; SEQ ID NO:87; SEQ ID NO:94; SEQ ID NO:106; SEQ ID NO:117; SEQ ID NO:130; SEQ ID NO:145, and SEQ ID NO:146, or a unique fragment thereof. Nucleic acid samples from collected mosquito larvae that specifically hybridize with such a probe under relatively high-stringency conditions (e.g. low salt conditions and/or high temperatures of hybridization) can be processed for the isolation of a mosquito-infecting baculovirus of this invention according to the procedures described herein.
As is known in the art, annealing reactions (between primers or probes and the sample DNA or RNA) are affected by the concentration, sequence complexity, base composition and length of the primer/probe; the concentration of monovalent cations; the presence of hybrid destabilizing agents (e.g. formamide); and the incubation temperature. The following formula relates many of these parameters to the Tm (the temperature at which half of the DNA molecules have dissociated into single strands): Tm=81.5xc2x0 C.+16.6 log M+41 (mole fraction of G+C)xe2x88x92(500/L)xe2x88x920.62 (% formamide), where M is the molar concentration of monovalent cations and L is the molecular length of the probe DNA in bases. Increasing the salt concentration from 100 mM to 1 M increases the Tm by 16xc2x0 C. Annealing temperatures that are 25xc2x0 C. below the Tm of the native DNA usually provide the maximum rate of hybridization. For example, for genomic DNAs, the annealing temperature is typically 65xc2x0 C. in aqueous solvents containing 250 mM Na+. Adding formamide to a final concentration of 50% lowers the annealing temperature to 42xc2x0 C. In general, the higher the probe concentration, the more rapid the annealing reaction. However, the competing reaction of probe strands reannealing to each other will also occur more rapidly. For this reason, probe concentrations need to be kept relatively low (xe2x89xa610 ng/ml hybridization solution). Alternatively, single-stranded probes can be used.
Stringency, or the degree to which mismatches are permitted in the binding of two single strands, is a critical parameter in all annealing reactions and is affected by salt concentration and annealing temperature. The Tm of a duplex decreases by approximately 1xc2x0 C. for each 1% of mismatched base pairs, except for short DNA (15-30 bases, for which each mismatch can reduce the Tm by 5xc2x0 C. (Wolff. R and Gemmill. R 1997 Purify and analyzing genomic DNA IN: Genome Analysisxe2x80x94A Laboratory Manual, Vol. 1; Birren, B et al., eds., Cold Spring Harbor Laboratory Press, Plainview, N.Y.). The higher the salt concentration, the greater the number of mismatches that can be tolerated at a given temperature. The final stringency of the reaction can then be readily adjusted by using a series of post-hybridization washes of increasing stringency. The specific hybridization signal should be assessed by autoradiography between these washes and compared with the background.
Several standard hybridization conditions have been developed on the basis of the considerations above. Stringent hybridization can be performed in an aqueous hybridization solution containing 2xc3x97SSC at 65xc2x0 C. General methods for optimizing and performing hybridizations are set forth in Sambrook et al., xe2x80x9cMolecular Cloning, a Laboratory Manual,xe2x80x9d Cold Spring Harbor Laboratory Press (1989). Any viruses so isolated can be confirmed to be a mosquito-infecting baculovirus of this invention by performing the biochemical/molecular, microscopic and infectivity characterizations described herein.
The novel baculoviruses of this invention can be engineered to express an insect-antagonistic gene under the control of a baculoviral promoter in amounts sufficient to produce a debilitating effect in the mosquito, for example paralysis or premature molting.
In one embodiment, a recombinant mosquito baculovirus can be constructed by inserting the promoter/insect-antagonistic gene at a location in the mosquito baculovirus genome that is a nonessential region for viral replication and propagation, by methods known to those skilled in the art. For example, the baculoviral genes identified as EGT, p10, DA26, ETL, p94 and orf603 have been shown previously to be good sites for insertion of an engineered promoter/insect-antagonistic gene construct. The egt gene in baculoviruses is typically located adjacent to the late expression factor 1 (lef-1) gene (SEQ ID NO:147, also present at nucleotides 2630-3334 in SEQ ID NO:13). The p10 gene is typically located immediately upstream of and in opposite orientation to the p74 gene (SEQ ID NO:148; also present at nucleotides 222-1253 in SEQ ID NO:66).
In a specific embodiment, the promoter/insect-antagonistic gene is inserted into the non-essential gene locus, by first constructing a transplacement, or transfer, plasmid which contains the desired promoter/insect-antagonistic gene flanked by CuniNPV non-essential gene sequences, and all the elements necessary for autonomous replication in bacteria, such as E. coli. Such plasmids are then cotransfectecd into insect cells or larvae with the transplacement plasmid and wild type CuniNPV, thus allowing the allelic replacement of the non-essential gene of CuniNPV with the desired promoter/insect-antagonistic gene constuct. The desired recombinant CuniNPV is isolated after selection on the basis of its non-essential gene function-negative phenotype and screening for the proper allelic replacement events by restriction endonuclease analysis and Southern hybridization. (Miller et al. (1987) xe2x80x9cBiotechnology in Invertebrate Pathology and Cell Culturexe2x80x9d pp. 295-303; Miller et al. (1986) in Genetic Engineering. Principles and Methods, Vol. 8, eds. J. Setlow and A. Hollaender, Plenum Press, New York, pp.277-298; U.S. Pat. No. 5,266,317).
The present invention also provides methods for controlling baculovirus infections in invertebrates such as beneficial insects and members of the Crustacea, e.g. shrimp species such as Penaeus duorarum and Penaeus aztecus. The baculovirus known as xe2x80x9cBaculovirus penaeixe2x80x9d, commonly known as xe2x80x9cBPxe2x80x9d and designated as PvSNPV (Francki, R. I. B., Fauquet, C. M., Knudson, D. L. and Brown, F. (Eds.) 1991 xe2x80x9cClassification and Nomenclature of Viruses,xe2x80x9d 5th Report of the International Committee on Taxonomy of Viruses; Springer-Verlag, New York) is known to infect at least 14 species of penaeid shrimp and can can cause serious epizootics with high mortality of larval and postlarval stages in some of those species (Lightner, D V and Redman R M 1991 Host, geographic range and diagnosis procedures for the penaeid virus diseases of concern to shrimp culturists in the Americas; In xe2x80x9cFrontiers in Shrimp researchxe2x80x9d; P. Deloach, W. J. Dougherty, and M. A. Davidson. eds., pp. 177-196; Elsevier, Amsterdam). BP is responsible for annual economic losses in cultured penaeid shrimp in the Americas and Hawaii due to mass mortalities that occur in the hatchery phase of production (Stuck, K C and Overstreet R M 1994 J Invertebr. Pathol 64:18-25). BP also commonly infects a substantial number of wild brown shrimp, P. aztecus, from the northern Gulf of Mexico (Overstreet, R M 1994 xe2x80x9cBP in Penaeid Shrimpsxe2x80x9d In USMSFP 10th Anniversary Review, Gulf Coast Research Laboratory Special Publ. No. 1:97-106). The method disclosed herein comprises administering to the invertebrate an amount of calcium, iron or copper ions that is effective in reducing or eliminating baculoviral infections. In specific embodiments, the effective calcium, iron or copper ion concentration is 1 mM, 5 mM or 10 mM. In a specific embodiment, calcium, iron or copper ions are added to the water in which shrimp are living or being farmed to give an effective concentration of at least 1 mM.
The invention also provides an isolated nucleic acid comprising the nucleotide sequence set forth in SEQ ID NO:1; SEQ ID NO:4; SEQ ID NO:10; SEQ ID NO:19; SEQ ID NO:38; SEQ ID NO:42; SEQ ID NO:44; SEQ ID NO:46; SEQ ID NO:48; SEQ ID NO:50; SEQ ID NO:53; SEQ ID NO:61; SEQ ID NO:66; SEQ ID NO:70; SEQ ID NO:72; SEQ ID NO:74; SEQ ID NO:76; SEQ ID NO:80; SEQ ID NO:83; SEQ ID NO:87; SEQ ID NO:94; SEQ ID NO:106; SEQ ID NO:117; SEQ ID NO:130; SEQ ID NO:145, and SEQ ID NO:146, as well as allelic variants, homologs or unique fragments thereof. To be unique, the fragment must be of sufficient size to distinguish it from other known nucleic acid sequences, most readily determined by comparing any nucleic acid fragment to the nucleotide sequences of nucleic acids in computer databases of such sequences, such as GenBank. Such comparative searches are standard in the art. The fragment may code for a polypeptide, e.g. SEQ ID NO:147 or SEQ ID NO:148, or it may be a primer or probe sequence. Typically, a unique fragment useful as a primer or probe will be at least about 20 to about 25 nucleotides in length, depending upon the specific nucleotide content of the sequence. Additionally, fragments can be, for example, at least about 30, 40, 50, 75, 100, 200 or 500 nucleotides in length. The nucleic acids can be single or double stranded, and can be DNA or RNA, depending upon the purpose for which it is intended.
The present invention additionally provides an isolated nucleic acid that hybridizes under stringent conditions with a nucleic acid selected from the group of sequences set forth in SEQ ID NO:1; SEQ ID NO:4; SEQ ID NO:10; SEQ ID NO:19; SEQ ID NO:38; SEQ ID NO:42; SEQ ID NO:44; SEQ ID NO:46; SEQ ID NO:48; SEQ ID NO:50; SEQ ID NO:53; SEQ ID NO:61; SEQ ID NO:66; SEQ ID NO:70; SEQ ID NO:72; SEQ ID NO:74; SEQ ID NO:76; SEQ ID NO:80; SEQ ID NO:83; SEQ ID NO:87; SEQ ID NO:94; SEQ ID NO:106; SEQ ID NO:117; SEQ ID NO:130; SEQ ID NO:145, SEQ ID NO:146, SEQ ID NO:147, and SEQ ID NO:148. In a specific embodiment, stringent conditions would include an aqueous hybridization solution comprising the nucleic acid probe and 0.07 M NaH2PO4, 0.18 M Na2HPO4, 0.001 M EDTA, 7% SDS, 1% BSA at 65xc2x0 C., followed by posthybridization washes as follows: 2xc3x97SSC/0.1%SDS at room temperature for 5 minutes, followed by two washes in 0.1xc3x97SSC/0.1%SDS at 65xc2x0 C. for 20 minutes each. Such hybridizing nucleic acids can be fragments of the complementary strands for the referenced sequences or can be primers or probes that can identify the referenced sequences.
In a specific embodiment, the invention provides an isolated nucleic acid encoding a polypeptide comprising the amino acid sequence set forth in SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:30, or SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:67, SEQ ID NO:69, SEQ ID NO:71, SEQ ID NO:73, SEQ ID NO:75, SEQ ID NO:77, SEQ ID NO:79, SEQ ID NO:81, SEQ ID NO:82, SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:91, SEQ ID NO:93, SEQ ID NO:95, SEQ ID NO:96, SEQ ID NO:97, SEQ ID NO:98, SEQ ID NO:99, SEQ ID NO:10, SEQ ID NO:102, SEQ ID NO:103, SEQ ID NO:104, SEQ ID NO:105, SEQ ID NO:107, SEQ ID NO:108, SEQ ID NO:109, SEQ ID NO:110, SEQ ID NO:111, SEQ ID NO:112, SEQ ID NO:114, SEQ ID NO:115, SEQ ID NO:116, SEQ ID NO:118, SEQ ID NO:119, SEQ ID NO:120, SEQ ID NO:121, SEQ ID NO:122, SEQ ID NO:123, SEQ ID NO:124, SEQ ID NO:125, SEQ ID NO:127, SEQ ID NO:128, SEQ ID NO:129, SEQ ID NO:131, SEQ ID NO:132, SEQ ID NO:33, SEQ ID NO:134, SEQ ID NO:135, SEQ ID NO:136, SEQ ID NO:138, SEQ ID NO:139, SEQ ID NO:140, SEQ ID NO:141, SEQ ID NO:142, SEQ ID NO:143, or SEQ ID NO:144. Homologies of these amino acid sequences to sequences in the computer databases are presented in Tables 11 and 12. Table 11 presents a comparison of these CuniNPV amino acid sequences with known Lepidopteran baculovirus amino acid sequences. Table 12 presents non-baculoviral sequence matches to the CuniNPV amino acid sequences in the databases. The analyses presented in both tables were done using BLASTP 2.0.8 (Altschul, S F et al 1997 Nucleic Acids Res. 25:3389-3402).
The present invention additionally provides a purified polypeptide or protein encoded by a nucleic acid of this invention. Purified means substantially free from the naturally occurring materials with which the polynucleotide or polypeptide is normally associated in nature. The purified protein need not be homogeneous, but must be sufficiently free of contaminants to be useful in research or commercial applications, for example, for use in detecting or preparing antibodies to the protein, or in screening libraries of molecules for those molecules that interact with the protein. Greater levels of purity can be obtained using methods derived from well known protocols. The polypeptide or protein can be readily obtained by any of several means. For example, the nucleotide sequence encoding the polypeptide can be translated and then the corresponding polypeptide, or any portion thereof, can be synthesized mechanically by standard methods. Additionally, the nucleic acids encoding the polypeptide can be expressed or synthesized, an antibody specific for the resulting polypeptide can be raised by standard methods (see, e.g., Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. 1988), and the polypeptide can be isolated from infected mosquito larvae or cells by selective binding with the antibody. Such polypeptides can be purified to the extent desired by standard methods of protein purification (see, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1989). The antibodies so produced can be used as diagnostic tools for the presence of the virus.
As will be appreciated by those skilled in the art, the invention also includes those polypeptides having slight variations in amino acid sequences or other properties. Amino acid substitutions can be selected by known parameters to be neutral (see. e.g., Robinson W E Jr. and Mitchell W M., AIDS 4:S151-S162(1990)). Such variations may arise naturally as allelic variations (e.g., due to genetic polymorphism) or may be produced by human intervention (e.g., by mutagenesis of cloned DNA sequences). Such as induced point, deletion, insertion and substitution mutants. Minor changes in amino acid sequence are generally preferred, such as conservative amino acid replacements, small internal deletions or insertions, and additions or deletions at the ends of the molecules. Substitutions may be designed based on, for example, the model of Dayhoff, el al. (in Atlas of Protein Sequence and Structure 1978, Nat""l Biomed. Res. Found., Washington, D.C.). These modifications can result in changes in the amino acid sequence, provide silent mutations, modify a restriction site, or provide other specific mutations. Likewise, such amino acid changes result in a different nucleic acid encoding the polypeptides and proteins. Thus, alternative nucleic acids are also contemplated by such modifications.
The present invention also provides cells containing a nucleic acid of the invention. A cell containing a nucleic acid encoding a polypeptide typically can replicate the DNA and, further, typically can express the encoded polypeptide. The cell can be a prokaryotic cell, particularly for the purpose of producing quantities of the nucleic acid, or a eukaryotic cell, particularly an insect cell. The cell is preferably an insect cell for the purpose of expressing the encoded polypeptide so that the resultant produced polypeptide has insect cell-determined protein processing modifications.
Throughout this application, various publications are referenced. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art to which this invention pertains.